Researchers from the Hebrew University of Jerusalem, Israel (HUJI), and the Technical University of Denmark (DTU) have used a nanophotonic chip etched with grooved aluminum plasmonic waveguides to detect a fluorescent signal from a single bacterial cell (Nano Lett., doi: 10.1021/acs.nanolett.7b02132). The team believes that the creation of this plasmonic-waveguide platform offers a route to creating compact, “hybrid bioplasmonic systems” that use live cells as optical beacons for reporting chemical or biological toxins.

Toxicity reporters

The prospect of using bacteria genetically modified to emit light as “signal flares” for specific toxic chemical compounds has long attracted interest. Under the scheme, a strain of a bacterium such as E. coli is engineered with an additional sliver of DNA that produces green fluorescent protein (GFP), or some other fluorescent or bioluminescent agent, when the bacterium encounters a particular toxic substance. Illuminating the bacteria with lasers to excite the fluorescence then allows researchers to determine whether the bugs have been exposed to the substance of interest.

The problem has been scaling these schemes down to a useful size. In general, sensing the faint optical signals from these biological reporters has required off-chip detection and a “cumbersome apparatus,” according the HUJI/DTU team. That makes it difficult to create “lab on a chip” and other compact devices that can be deployed in the field.

Better than gold

To get at a chip-scale device, the HUJI researchers, led by OSA Fellow Uriel Levy, worked with OSA life member Anders Kristensen of DTU to develop an approach that leveraged plasmonics to boost the signal from fluorescent bacteria. Specifically, they settled on a chip that included a V-shaped, grooved plasmonic waveguide 10 to 30 microns long and around 3 microns wide at the top, tapering down in a V cross-section to a few tens of nanometers in width at the bottom. Such waveguides have emerged in the past few years as strong structures for amplifying and channeling optical signals in nanoscale sensors.

In this specific study, Levy and Kristensen reasoned, the V-shaped waveguides could do triple duty, serving to mechanically trap individual bacteria, plasmonically amplify the optical signal from them, and channel the signal to an output nanocoupler at the end of the waveguide for detection. Interestingly, while many plasmonic structures rely on noble metals like gold or silver, the chip fashioned by the HUJI/DTU team used a much more common substance, aluminum. That's a choice, according to the team, that’s both cost-effective and meshes well with the plasmonic modes in the short visible range that are most relevant for fluorescent proteins.

Engineered E. coli

To test the system, the team turned to Shimshon Belkin of the HUJI Institute of Life Sciences to engineer an appropriate bug. Belkin created a strain of E. coli that included a plasmid that would churn out GFP in the presence of DNA-damaging chemicals such as nalidixic acid (NA). The researchers then exposed the bacteria to NA, and tested the chemically exposed bacteria by cycling them through a flow cell containing the waveguide and exposing them to a beam from a 488-nm argon laser to excite fluorescence.

The researchers found that, as individual bacteria flowed in solution above the waveguide, the plasmonic coupling of the fluorescence in the waveguide was sufficiently strong to result in a good signal at the nanocoupler ends. Further, the team was able to use the laser to directly stimulate a single bacterial cell that had become mechanically trapped in the waveguide (see image above); again, the researchers were able to collect significant fluorescence at the output nanocoupler. Taken together, the team concludes, the experiments showed that the setup allowed a bright signal to be harvested from a single bacterium—whether in whether in “wet” or “dry” conditions.

Toward a field-ready system

Belkin, who worked on engineering the strain of E. coli used in the experiments, noted in an email to OPN that the bugs were designed to create GFP in response to any DNA damage—not just to damage attributable to nalidixic acid, the specific chemical used in these tests. Instead, he writes, the response of the sensor strain is “very broad and general.” It’s also dose dependent, as “the more potent the DNA damage potential, the stronger is the fluorescence.” That suggests, says Belkin, that these sensor strains could be useful in responding to a variety of toxic agents in air, water, food and other substances.

The team believes that, by leveraging those sensitive bacterial beacons, the plasmonic-waveguide platform sets the stage for integrated, hybrid bioplasmonic sensors that could be feasible for field use. Such systems, says team leader Levy, could include implementations like specialized biochips for on-site detection of toxic chemicals in food, or even online chips that could provide “real-time warning against the presence of hazardous chemicals in drinking water systems.”

A typical chip-based system, he continues, might include a laser or LED light source coupled to a network of waveguides on the chip. In this scheme, each waveguide might contain a bacterium engineered to fluoresce in response to a different chemical or biological agent. An on-chip detector to pick up the fluorescent signal would complete the system.

A next step to get there, according to the team, will be the construction of such waveguide networks, and finding ways to immobilize the right bugs in the waveguides. Levy notes that techniques already used in creating DNA biochips could be leveraged to deposit bacteria in just such a patterned fashion. That, he concludes, could set up “the possibility of generating arrays of different sensor strains, capable of multiplexed detection of diverse chemical threats.”